This program is focused on the study of the enzyme systems for the biosynthesis, oxidative decarbosylation, and other related metabolic pathways of brain 2-hydroxy fatty acids. During the past three years, we have for the first time demonstrated and partially characterized the system for in vitro synthesis of brain cerebronic acid (2-hydroxytetracosanoic acid) from lignoceric acid (tetracosanoic acid). We found that the enzyme activity involves three components of brain, mitochondrial and two water-soluble factors (one heat-labile and the other heat-stable), and is stimulated further by microsomes. We will purify these components for a better understanding of the alpha-hydroxylation system. We also obtained much evidence which indicates that the alpha-hydroxylation may be the rate-limiting step of cerebroside biosynthesis and is closely related with myelin formation. The alpha-hydroxylation system appears to be closely integrated with the system of cerebroside synthesis, and all newly synthesized cerebronic acid is found not as free cerebronic acid but as a component of ceramide and cerebroside. In this respect, we propose studying the enzymic systems which may be involved in transferring the newly synthesized cerebronic acid to these sphingolipids in brain in order to further understand the control mechanism of 2-hydroxy fatty acid metabolism. We also demonstrated that the defective activity in mitochondria of myelin-deficient mouse mutant brain can be partially restored by the addition of microsomes from normal mice. Further investigation in this direction, by reconstituting subcellular fractions of the enzyme system from various sources of tissues and animal species is proposed. A new and simpler assay system for alpha-hydroxylation by using (R)-(2-3H)lignoceric acid as well as a new assay for the oxidation of 2-hydroxy fatty acid to 2-keto acids by measuring the reverse reaction is described.